SUMMARY The human microbiome is important for infectious disease pathogenesis. However, our understanding of the microbiome?s role in tuberculosis (TB), which is arguably the most important lung disease in the world, is extremely limited. In sub-Saharan Africa, TB is exacerbated by HIV which, even with antiretroviral therapy, results in reduced pulmonary immunity. The site-of-disease in active TB (bronchoalveolar space) is a unique environmental and immunological niche but its microbiota is surprisingly understudied. We do not know how taxa, including those important for lung heath (oral anaerobic fermenters), correlate with bacterial fermentation end-products like short chain fatty acids (SCFAs), which may influence immunological control of TB and tissue repair. Furthermore, the TB regimen is comprised of thousands of doses of antibiotics yet its long-term effect on the lung microbiota is hitherto uncharacterized. We hence lack key foundational knowledge that precludes research on the lung microbiota as a potential diagnostic or therapeutic target to improve TB outcomes. We will test our central hypothesis that site-of-disease oral anaerobic fermenters are associated with elevated pulmonary SCFAs and impaired inflammation and tissue repair biomarkers in TB cases (n=50) and, at treatment end, these taxa and biomarkers remain perturbed but improve a year later. We will recruit an equal number of HIV-positive patients at our high TB-HIV burden site in Cape Town. We will test our central hypothesis using three aims. Aim 1 will, using a modified bronchoalveolar lavage (BAL) procedure, compare the site-of-disease microbiota to that in contralateral non-diseased lung tissue before treatment. Aim 2 will characterize, at each lung site before treatment, the association between specific taxa, SCFAs, inflammation and tissue repair biomarkers, and investigate whether SCFA addition to ex vivo stimulated BAL cells impairs immune marker release in a dose-dependent manner. Aim 3 will re-sample patients by bronchoscopy at treatment end and a year later, and repeat measurements of the microbiota, SCFAs, and host biomarkers at each lung site. If the site-of-disease is associated with a perturbed microbiota, linked via SCFAs, to impaired pulmonary immunity and tissue repair, including after treatment, it will justify study of the microbiota and long-term TB clinical outcomes (e.g., progression, treatment failure, relapse), which requires large and expensive trials. It will enable research on tests or therapeutic interventions (antibiotics, drugs, prebiotics, vaccines) that target the microbiota. Key to achieving our aims are the transfer of the modified bronchoscopy and BAL microbiota sampling procedure (required to minimize cross contamination in low microbial abundance lower airway specimens) and leading-edge computational expertise (required to co-analyze sequence data in conjunction with biomarker and clinical data) from New York University to Stellenbosch University (SU). South African clinicians and scientists will train in each area by through research and training visits with the long-term aim of establishing a research program on the lung microbiota and respiratory health at SU.